Our previous studies have demonstrated the oxidative stress properties of
sodium ascorbate (SAA) and its
benzaldehyde derivative (SBA) on
cancer cell lines, but the molecular mechanisms mediating their cytotoxicity remain unclear. In this study, we treated human
colon cancer HT-29 cells with SAA and SBA, and found a significant exposure time-dependent increase of cytotoxicity in both treatments, with a higher cytotoxicity for 24 h with SAA (IC(50) = 5 mM) than SBA (IC(50) = 10 mM). A short-term treatment of cells with 10 mM SAA for 2 h revealed a destabilization of the lysosomes and subsequent induction of cell death, whereas 10 mM SBA triggered a remarkable production of reactive oxidative species, phosphorylation of survival
kinase AKT, expression of
cyclin kinase-dependent inhibitor p21, and induction of transient growth arrest. The crucial role of p21 mediating this cytotoxicity was confirmed by isogenic derivatives of the human colon
carcinoma HCT116 cell lines (p21(+/+) and p21(-/-)), and immunoprecipitation studies with p21 antibody. The SAA cytotoxicity was blocked by co-incubation with
catalase, whereas the SBA cytotoxicity and its subsequent growth arrest were abolished by
N-acetyl-L-cysteine (NAC), but was not affected by PI3K phosphorylation inhibitor
LY294002, or
catalase, suggesting two separated oxidative stress pathways were mediated by these two ascorbates. In addition, neither active
caspase 3 nor apoptotic bodies but autophagic vacuoles associated with increased LC3-II were found in SBA-treated HT-29 cells; implicating that SBA induced AKT phosphorylation-autophagy and p21-growth arrest in
colon cancer HT-29 cells through an NAC-inhibitable oxidative stress pathway.